The goal of the project is to understand the detailed molecular mechanism of HIV-1 DNA integration, the structures of the nucleoprotein complexes that mediate DNA integration, the mechanism of action of integrase inhibitors, and how the virus can the virus can evolve resistance to these inhibitors. Integration of a DNA copy of the viral genome into cellular DNA is an essential step for replication of HIV-1 and other retroviruses. Integration is mediated by the virally encoded integrase protein in complex with viral and target DNA; complexes of integrase associated with a pair of viral DNA are collectively called intasomes. The first intasome on the integration reaction pathway is Stable Synaptic Complex (SSC) intasome that comprises a complex of integrase and the pair of viral DNA ends. Integrase cleaves two nucleotides from each 3' end of the viral DNA (3' end processing) within the SSC and then integrates these 3' ends into target DNA (DNA strand transfer) to form the Strand Transfer Complex (STC) intasome. The FDA has recently approved three drugs, Raltegravir, Elvitegravir and Dolutegravir, that target HIV-1 integrase and more are in the pipeline. These drugs are highly effective and provide a new class of drugs for combination antiviral therapy. They specifically target the DNA strand transfer step of integration and bind to the assembled SSC intasomes after 3 end processing rather than free integrase protein. High-resolution structural studies of HIV-1 intasomes are therefore required to understand the detailed mechanism of action of inhibitors and mechanisms of escape by mutations that confer resistance. We have established conditions for in vitro assembly of HIV intasomes. The intasomes assembled in vitro mimic all the properties of the association of integrase with viral DNA in preintegration complexes (PICs) isolated from virus-infected cells. Structural studies of HIV intasomes have been frustrated by aggregation of both integrase and intasomes. We have recently overcome these obstacles. Fusing of Sulfolobus solfataricus chromosomal protein (PDB: 1BNZ) to the N-terminus of HIV-1 integrase resulted in a hyperactive protein that assembled intasomes with improved solubility properties. We have also assembled intasomes for our structural studies on branched product DNA, a strategy we have previously validated with the closely related prototype foamy virus integrase. Although the intasomes appeared to be homogeneous as judged by gel filtration, attempts to crystallize were unsuccessful. We there initiated collaboration with Dmitry Lyumkis at the Salk Institute to determine their structure by cryo-EM. The small size of HIV intasomes and the requirement for a high-ionic strength buffer containing glycerol present changes for cryo-EM. Nevertheless, we have obtained cryo-EM structures of HIV STC intasomes with a resolution ranging from 3.5 Angstroms near the core of the intasome to 4.5 Angstroms in peripheral regions. The overall structure is tetrameric and similar to the previously reported PFV intasome structures. The two inner subunits in the tetramer are mainly responsible for interactions with DNA. The C-terminal domains o the other subunits contribute to interactions with viral DNA, while the N-terminal domains of the outer subunits are disordered. In addition to the tetrameric intasomes, there is also a population of higher-order STC intasomes. The best resolved of the higher order intasomes is dodecameric. The dodecameric STC intasome has the same set of positionally conserved domains interacting with DNA, but the subunits to which they belong differ. The positionally conserved domains that interact with DNA in both the tetrameric and higher order intasomes are shared in common with intasomes of other retroviruses. Our STC structures were solved using HIV-1 integrase with an N-terminal fusion of the Sso7d domain. The multiple intasome species we observed, including tetrameric and dodecameric, raises the question as to whether the Sso7d domain influences the intasome structures. We have therefore also carried out structural studies of intasomes formed with wildtype HIV-1 integrase. Although the biophysical properties of wildtype HIV-1 integrase do not allow high resolution structural studies, we have obtained low resolution structures of wild type HIV-1 intasomes by negative staining and cryo-EM. As with the Sso7d fusion protein, multiple intasome species are observed, including tetrameric, octameric and decametric. We conclude that the fusion domain does not significantly influence the intasome structures. In addition to discrete intasomes, we also observed chains of heterogeneous length that result from domain swapping among intasomes. This is the structural basis of the aggregation problem that has hindered structural studies and we are pursuing strategies to overcome this problem Currently approved drugs that inhibit HIV DNA integration bind to Cleaved Stable Synaptic Complex (cSSC) intasomes. Having successfully determined structures of STC intasomes, our focus is now structural studies of cSSC intasomes. We have successfully devised strategies to assemble cSSC intasomes and their structures in complex with integrase inhibitors are being studies by cryo-EM in collaboration with Dmitry Lyumkis at the Salk Institute. Thus, we now have a platform for directly determining the structures of HIV intasomes in complex with inhibitors and studying the molecular mechanisms of drug resistance.